野油菜黄单胞菌8004甘天丝亮特征序列酯酶及其酯酶结构域在大肠杆菌中的表达,包涵体复性及性质(英文)  

Glycine-aspartic_ acid-serine-leucine esterase Xcc_ est from Xanthomonas campestris pv.campestris 8004 and its esterase domain:gene expression in Escherichia coli,refolding and characterization

作　　者：王建军[1] 杨柳[1] 曹燕萍[2] 郑国钧[2] 

机构地区：[1]微生物资源国家重点实验室,中国科学院微生物研究所,北京100101 [2]北京化工大学制药工程系,北京100029

出　　处：《微生物学报》2009年第2期191-197,共7页Acta Microbiologica Sinica

基　　金：Supported by the National Programs for High Technology Research and Development of China(2006AA02Z250);the Key Project of China NationalPrograms for Fundamental Research and Development of the Ministry of Science and Technology of China(2004CB719606)~~

摘　　要：【目的】了解野油菜黄单胞菌(Xanthomonas campestris pv.campestris)8004 GDSL(蛋白序列中甘氨酸、天冬氨酸、丝氨酸和亮氨酸特征序列)酯酶的性质。【方法】利用PCR方法扩增Xcc_ est及其不同结构域的基因,这些基因以组氨酸标签融合蛋白的形式在大肠杆菌中获得表达。融合蛋白通过镍亲和色谱纯化。【结果】部分纯化的Xcc_ est在催化对硝基苯丁酸酯时,最适pH值为8.0 ,最适温度为52℃。Xcc_ est对于对硝基苯丁酸酯的Km值和Vmax值分别是47.6 ±4.6μmol/L,和67.6 ±7.8 U/mg,Xcc_ est的酯酶结构域(Xcc_ est N1-334)对于同一底物的Km值和Vmax值分别是469.4 ±9.8μmol/L和2.5 ±0.9 U/mg。Xcc_ est的成熟结构域(Xcc_ est N26-606)可以获得成功复性,但是成熟酯酶结构域(Xcc_ est N26-334)不能获得复性。复性后的Xcc_ est N26-606底物谱较广,在室温下具有较高稳定性。【结论】复性的成熟结构域蛋白(Xcc_ est N26-606)具有一定的生物转化应用前景。[Objective]To characterize the GDSL （glycine, aspartic acid, serine and leucine motif in protein sequence） esterase Xcc_ est from Xanthomonas campestris pv. campestris （Xcc） 8004. [Methods] Xcc_ est gene and different domains of Xcc_ est gene were PCR amplified and expressed in Escherichia coli, the HIS-Tagged fusion proteins were purified by Ni-NTA chromatography. [Results] The optimum pH and temperature of partly purified Xcc_ est were 8.0 and 52℃ when pNPB （4-nitrophenylbutyrate） was used as substrate. The K_m and V_max value of Xcc_ est and the passenger domain （Xcc_ estN1-334） for pNPB were 47.6±4.6 mol/L, 67.6±7.8 U/mg and 469.4±9.8 mol/L, 2.5±0.9 U/mg respectively. Inclusion bodies of mature domain Xcc_ est （Xcc_ estN26-606） could be refolded but inclusion bodies of the passenger domain （Xcc_ estN26-334） could not be refolded. Refolded mature domain had broad substrate spectrum and showed higher stability than Xcc_ est when stored at 25℃. [Conclusions] Refolded Xcc_ estN26-606 can be a candidate for biotransformation application.

关 键 词：GDSL酯酶 野油菜黄单胞菌(Xanthomonas campestris) 复性 

分 类 号：Q78[生物学—分子生物学]